ABSTRACT Development of a preventive AIDS vaccine is a daunting task given the structural complexity of the HIV-1 envelope (Env) protein as well as extensive antigenic variation among viral quasispecies driven by immune escape mechanisms. Moreover, immunodominance toward non-neutralizing epitopes on the Env trimer, the sole viral antigen on the virion surface, makes the elicitation of broadly neutralizing antibody (bnAb) by vaccination particularly difficult. Nonetheless, immunological and structural characterizations of isolated bnAbs from HIV-1 infected donors have guided additional neutralizing target epitopes along with new approaches in vaccine strategies. The membrane proximal external region (MPER) of gp41 subunit is an attractive bnAb target given its linear and conserved epitope sequences as exemplified by 4E10, 10E8 and DH511 and 2F5 mAbs. However, MPER immunogen vaccines including peptides, protein scaffolds or MPER/liposomes have all failed to elicit neutralizing activities, suggesting incomplete mimicry of the quaternary structure on the virion surface. Furthermore, the accessibility of the MPER is limited, being shielded by gp160 trimer ectodomain from above and the viral membrane from below, contributing to the poor immunogenicity of the MPER elicited by trimer immunogens. A closed rather than open configuration of gp140 trimer immunogens has proven to be important for elicitation of bnAbs directed to epitopes in gp120. Likewise, our recent MPER/liposome results suggest that the unrestricted approach angle afforded to the B cell receptor with current vaccine formulation is problematic, resulting in induction of a majority of Abs without neutralizing activity or gp160 trimer reactivity. Therefore, MPER immunogens must mimic spatial occlusion and enforce limited Ab accessibility to the MPER in a manner analogous to that imposed by the quaternary structural configuration of gp160 on the virion surface. Given sequence variations in the N-terminal region of the MPER, we will develop strategies to augment subdominant Ab responses directed to the MPER C-terminal region in Aim 1. A knock-in (KI) mouse model generating antibodies with long CDRH3 loops, extrinsic factors such as cyclic di-GMP adjuvant, ICOSL and persistent antigen supply will be tested independently or in combination for their impact on augmenting MPER C-terminal region-specific Abs. In Aim 2, we shall exploit nanodisc technology that serves as a platform for the assembly of gp160 into a native membrane-like environment to prime or boost MPER-specific Ab responses with a desirable approach angle and to eliminate off-target vector responses. In conjunction with optimized vaccine regimen in Aim 1, we shall pursue complementary heterologous immunization strategies in mouse and rabbit models to foster the induction of 4E10/10E8-like bnAbs. DNA C-particle and MPERTM/liposome immunogens will further disfavor expansion of gp120-41 directed dominant undesirable Ab responses elicited by the gp160/nanodisc, while facilitating the induction of sufficient serum titers of bnAbs with requisite approach angles. Genetic and biophysical features of MPER-specific bnAbs will be determined.